牛支原体新疆分离株重组P81膜蛋白的表达及鉴定  

An experiment to express and identify membrane protein P81 from Mycoplasma bovis isolates from Xinjiang

作　　者：赵阳 陈创夫 剡根强[2] 石峰[3] ZHAO Yang;CHEN Chuang-fu;YAN Gen-qiang;SHI Feng(Key Laboratory of Diseases Highly Prevalent in Xinjiang and Its Ethnic Groups, Xinjiang Production and Construction Corps, Xinjiang, Shihezi 832003;Laboratory of Infectious Veterinary Diseases ,College of Animal Science and Technology , Shihezi University;College of Life Science, Shihezi University)

机构地区：[1]新疆生产建设兵团西部地方与民族高发病重点实验室,新疆石河子832003 [2]石河子大学动物科技学院兽医传染病实验室 [3]石河子大学生命科学学院

出　　处：《中国病原生物学杂志》2017年第10期947-951,共5页Journal of Pathogen Biology

基　　金：国际合作项目:牛病毒性腹泻防治新技术的研究(No.KZ0020)

摘　　要：目的重组表达牛支原体P81膜蛋白,为研制牛支原体疾病诊断试剂提供研究基础。方法根据GenBank发表的牛支原体P81基因的编码序列,利用点突变试剂盒基于重叠延伸PCR原理设计5对引物,将P81基因的第165、690、1 455、1 674位编码色氨酸的TGA密码子分别突变为TGG,并连接到表达载体pET-32a(+),转化DH5α感受态细胞后经IPTG诱导表达目的蛋白,Western blot检测其反应原性。结果成功扩增出4处TGA突变为TGG的牛支原体新疆分离株P81基因,大小与预期值2 112bp一致。重组表达载体转化菌经IPTG 37℃诱导8h,表达分子质量单位(Mr)约94×103的目的蛋白,与预期相符。Western blot检测该蛋白能被抗P81多克隆抗体识别。结论成功构建重组牛支原体P81膜蛋白基因表达载体,获得的目的蛋白具有良好反应原性,为研发牛支原体病免疫诊断试剂奠定了基础。Objective To express membrane protein P81 of Mycoplasma bovis in a recombinant expression system to lay the foundation for the development of reagents to diagnose mycoplasmosis. Methods In accordance with the coding sequence of the P81 gene of M.bovis published in GenBank,a site-directed mutagenesis kit was used to design 4 pairs of primers based on overlap extension PCR.A mutation from TGA to TGG in tryptophan codons 165,690,1 455,and1 674 of the P81 gene.A fragment containing the mutation was ligated into the expression vector pET-32 a（＋）and transformed into DH5αcompetent cells.Expression of the target protein was induced with IPTG,and Western blotting was used to detect its reactivity. Results Fragments of the P81 gene with the 4 mutations from TGA to TGG were successfully amplified from M.bovis isolates from Xinjiang.The size of the gene was consistent with an expected size of2 112 bp.The recombinant expression vector was transformed into competent cells and expression of the target protein was induced with IPTG for 8 hat 37 ℃.The molecular mass（Mr）of the expressed target protein was about 94×103,which was in line with expectations.Western blot indicated that the protein was recognized by polyclonal antibodies against P81. Conclusion A gene vector was successfully constructed for expression of recombinant P81 membrane protein from M.bovis and the target protein was obtained.This protein is immunogenic,and this finding lays the foundation for the development of immunological reagents of diagnose M.bovis.

关 键 词：牛支原体新疆分离株 定点突变 P81基因 WESTERN BLOT 

分 类 号：R384.4[医药卫生—医学寄生虫学]